US5118606A - Methods for detecting cellular pathology by assaying spectrin and spectrin breakdown products - Google Patents

Methods for detecting cellular pathology by assaying spectrin and spectrin breakdown products Download PDF

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US5118606A
US5118606A US07/558,700 US55870090A US5118606A US 5118606 A US5118606 A US 5118606A US 55870090 A US55870090 A US 55870090A US 5118606 A US5118606 A US 5118606A
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spectrin
sample
breakdown products
total amount
degradation
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Gary S. Lynch
Peter A. Seubert
David D. Eveleth, Jr.
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University of California
University of California San Diego UCSD
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Assigned to REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE reassignment REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE ASSIGNMENT OF ASSIGNORS INTEREST. Assignors: EVELETH, DAVID D. JR.
Priority to DK91913322.3T priority patent/DK0540591T3/da
Priority to JP3512466A priority patent/JP2824150B2/ja
Priority to ES91913322T priority patent/ES2077863T3/es
Priority to PCT/US1991/004910 priority patent/WO1992001935A1/en
Priority to EP91913322A priority patent/EP0540591B1/de
Priority to CA002088050A priority patent/CA2088050A1/en
Priority to AT91913322T priority patent/ATE128233T1/de
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G01N33/561—Immunoelectrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/811—Test for named disease, body condition or organ function
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation

Definitions

  • This invention relates to an in vitro method for the detection of cellular pathology, and more specifically to an assay for monitoring cytoskeleton breakdown products to determine cell necrosis.
  • the structural integrity of cells is maintained in part by the cytoskeleton, a mesh-like structure composed primarily of proteins, which lies adjacent to the inner cell surface.
  • the cytoskeleton of many cell types (a partial list includes neurons, lymphocytes, kidney, liver, cardiac and smooth muscle, and blood platelets) contain large amounts of a protein either identical to or closely related to brain spectrin (also known as fodrin). Spectrin binds F-actin, and together they are generally associated with the inner face of the cell membrane, where they form a filamentous meshwork.
  • calpain calcium activated neutral protease
  • calpain I requires micromolar calcium
  • calpain II is activated by concentrations of calcium between 0 1 and 0.5 mM.
  • the two forms are differentially distributed in the brain. While calpain II is mainly localized in the cytoplasmic fraction of brain cells, the highest activity of calpain I is found in small processes. While the two forms of calpain differ in these and other ways, the term "Calpain" shall be used herein to refer to calcium activated neutral proteases generally, including both forms of calpain.
  • a variety of cellular insults e.g., toxins, anoxia, etc.
  • disease states e.g., Alzheimer's, Parkinson's, HIV-induced neuropathy, muscular dystrophy
  • Alzheimer's, Parkinson's, HIV-induced neuropathy, muscular dystrophy cause the degeneration and death of cells.
  • Preferably such methods also have high sensitivity, wide ranging applicability and ease of administration.
  • the present invention provides a method of detecting cellular death or degradation in a subject, such as a mammal, comprising analyzing a biological sample from the subject for the presence of spectrin breakdown products and comparing the quantity of spectrin breakdown products to the quantity of spectrin breakdown products in a normal subject, wherein an increased level of spectrin breakdown products indicates cellular death or degradation in the subject.
  • the quantity of spectrin breakdown products in the normal subject is substantially undetectable.
  • the biological sample can be any sample from an organism, especially cerebrospinal fluid or a component of blood.
  • the cellular death or degradation detected can, for example, be due to a non-pathological cellular insult, such as a trauma, ischemia, lesions, or exposure to toxins; or may be due to a pathology, including those of the nervous system, such as Alzheimer's disease, Parkinson's disease and muscular dystrophy.
  • Biological samples for the detection of cellular death or degradation in the nervous system can include neural tissue or cerebrospinal fluid.
  • the step of analyzing the biological sample for the presence of spectrin breakdown products can comprise, for example, contacting a spectrin breakdown product in the sample with a detectably labeled antibody, and can also include the steps of exposing the sample to an electrical gradient so as to separate the components in such a way that spectrin breakdown products are separated from spectrin, contacting the separated components with a detectably labeled antibody that binds to a spectrin breakdown product, and determining the presence of antibody binding, wherein the presence of the antibody binding indicates the presence of spectrin breakdown products.
  • the step of analyzing the biological sample can also comprise the step of staining a separated product with a stain which visualizes the product, and determining the presence of stain binding, wherein the presence of the stain indicates the presence of spectrin breakdown products.
  • a method of detecting cellular death or pathology in a sample from a subject comprising obtaining a biological sample from the subject, analyzing the biological sample for the presence of spectrin breakdown products, determining a basal level of spectrin breakdown products, and comparing the determined level to the basal level, wherein a higher level than the basal level indicates cellular death or degradation.
  • the basal level of this method is usually the basal level of spectrin breakdown products in a normal subject, and in many instances can be assumed to be zero.
  • the sample can be any biological sample from the subject, including cerebrospinal fluid, a tissue sample, or blood or any component of blood.
  • a method of detecting cellular death or degradation in a subject comprising obtaining a biological sample from the subject; determining the total amount of spectrin, including intact spectrin and spectrin breakdown products, in the sample; determining a basal quantity of the total amount of spectrin; and comparing the determined quantity of the total amount of spectrin to the basal quantity of the total amount of spectrin; wherein a quantity of the total amount of spectrin in the sample greater than the basal quantity indicates cellular death or degradation.
  • the total amount of spectrin can be measured as total spectrin immunoreactivity through such means as an ELISA assay or a Western Blot assay.
  • the sample can be any biological sample from the subject, including cerebrospinal fluid, a tissue sample, or blood or any component of blood.
  • Another aspect of the present invention provides a method of enriching a biological sample for spectrin breakdown products relative to intact spectrin in a biological sample, comprising precipitating intact spectrin in said sample, leaving spectrin breakdown products in solution in said sample by altering conditions which differentially affect the solubility of the intact spectrin molecule and the spectrin breakdown products; and collecting the precipitated or soluble spectrin breakdown products.
  • the step of precipitating intact spectrin or spectrin breakdown products can comprise altering the pH or ionic strength of the biological sample solution.
  • FIG. 1 A Spectrin immunoreactivity in blotted samples of the contralateral (left lane) and ipsilateral (right lane) dentate gyrus two days after a unilateral lesion of the entorhinal cortex.
  • Arrows indicate the alpha and beta spectrin subunits with apparent M r s of about 240 and 230 kilodaltons (“kD”), respectively, and two additional immunoreactive peptides (BDP1 and BDP2) with apparent M r s of about 155 and 150 kD, respectively
  • Lane 1 Purified brain spectrin incubated under the following conditions: Lane 1: no additions; Lane 2: 1 mM CaCl 2 , 1.8 ⁇ g/ml calpain I, 10 minutes; Lane 3: 1 mM CaCl 2 , 3 ⁇ g/ml calpain I, 30 minutes; Lane 4: 10 ⁇ g of dentate gyrus protein homogenate two days post-lesion; Lane 5; 1 mM CaCl 2 , 13 ⁇ g/ml calpain, 1.25 ⁇ g/ml calmodulin, 3 minutes; Lane 6: 1 mM CaCl 2 , 13 ⁇ g/ml calpain, 7.25 ⁇ g/ml calmodulin, 30 minutes.
  • FIG. 2 A Time course of the changes in BDP1 and BDP2 in the ipsilateral side of dentate gyrus following a unilateral entorhinal cortex lesion.
  • B Time course of the changes in BDP1 and BDP2 in the contralateral side.
  • BDP1 (filled circles) and BDP2 (open circles) levels are expressed as a percentage of the total spectrin immunoreactivity, as determined by scanning relative densitometry.
  • FIGS. 3 Levels of BDP's in regions of the brain of control and Brindled mice, showing the effect of treatment with copper.
  • FIG. 4 Levels of BDP's in the dentate gyrus and the CA1 region of the hippocampus of rats receiving trimethyltin.
  • FIG. 5 Levels of BDP's in the dentate gyrus and the CA1 region of the hippocampus from a gerbil following ischemia.
  • FIG. 6 Standard curve showing absorbance at 405 nm for various levels of spectrin in an ELISA assay.
  • FIG. 7 Left (rostral to caudal, 1-4) and right (5-8) hippocampi of a control animal and the left (9-12) and right (13-16) hippocampi from a kainate injected animal.
  • FIG. 7A shows the results of the Western blot assay.
  • FIG. 7B shows the results of the ELISA assay.
  • FIG. 8 Spectrin immunoreactivity of CSF samples, as measured by ELISA, from two patients of group 1 (unruptured aneurisms) and four patients of group 2 (ruptured aneurisms).
  • the present invention relates to sensitive and efficient methods for the early detection of cellular death and degradation.
  • the methods of the invention detect cellular death and/or degradation through an assay for spectrin or the breakdown products of spectrin.
  • Calpain The activation of Calpain leads to the proteolysis of many proteins including spectrin. Accordingly, Calpain is believed to cause production of spectrin breakdown products ("BDP's") from spectrin in dead and degenerating cells. Thus, the detection of BDP's is believed to advantageously serve as an indicator of the activation of Calpain.
  • BDP's spectrin breakdown products
  • BDP's are unusually stable polypeptides. BDP's can be detected in vivo for up to as long as two months or more after release from the cytoskeleton. Thus, BDP's can advantageously remain to serve as indicators of cellular death or degradation during this period of stability.
  • Calpain is an early event in cell death. This is in contrast to other known proteases which are believed to be activated only in the late stages of cell death. The activation of Calpain is believed to often occur before the onset of pathological symptoms associated with cell death. Thus, the detection of BDP's is believed to advantageously be useful as a method for the early detection of cell death, potentially prior to the onset of pathological symptoms.
  • the present invention advantageously provides a method for the detection of cellular pathology by means of an immunoassay to determine the presence of BDP's of spectrin.
  • Two major BDP's are known, BDP1 and BDP2.
  • the components from a sample of spectrin-containing cells are physically separated, as by exposure to an electric field, in such a way that BDP's and spectrin are separated.
  • the separated components can then be visualized, as by staining with a stain such as Coomassie Blue.
  • antibodies reactive with BDP's can then be contacted with the separated sample, and antibody binding to the portion of the sample containing BDP's determined.
  • the determined amount of BDP's can then be compared with a basal level of BDP's in similar samples from normal patients.
  • An increase in the level of BDP's is indicative of cellular death or degradation.
  • the basal level will be a level which is below the detection threshold of the methods herein described. Thus, in these cases, the detection of any immunoreactivity is an indication of cellular death or degradation.
  • the invention provides an additional method for the detection of cellular pathology to determine the presence of intact spectrin itself or spectrin immunoreactivity.
  • an enzyme-linked immunosorbent assay for spectrin immunoreactivity in biological samples, such as tissue extracts, cerebro-spinal fluid (CSF), or blood serum is provided.
  • ELISA enzyme-linked immunosorbent assay
  • One particular application of the method is the detection of spectrin or spectrin immunoreactivity in the CSF as an indication of neurodegenerative conditions such as subarachnoid hemorrhage, Alzheimer's Disease, HIV-induced neuropathy and/or stroke.
  • BDP's were identified from a sample of purified spectrin, by exposing the sample to Calpain for varying lengths of time and exposing the treated samples to SDS-PAGE.
  • Brain spectrin was purified to greater than 90% purity by the method of Davis and Bennett, J. Biol Chem. 258:7757-7766 (1983), which is incorporated herein by reference Calpain was purified to a similar level of purity from rat erythrocyte cytosol according to the method of Seubert, et al., Synapse 1:20-24 (1987), which is incorporated herein by reference.
  • Spectrin at a concentration of 75 ⁇ g/ml was incubated at 30° C. with 100 ⁇ M CaCl 2 , 3 ⁇ g/ml calpain I, 20 mM Tris-Cl, 5 mM ⁇ -mercaptoethanol and 150 mM NaCl at pH 7.5. Aliquots were withdrawn at 10 minutes and at 30 minutes. The aliquots were added to one-third volume of 3X SDS-PAGE buffer (150mM Tris-Po 4 , 6%, SDS, 30% glycerol, 3.75 mM EDTA, 3% ⁇ -mercaptoethanol, pH 6.8). The samples were heated in a 90° C.
  • the peptides of the two 150 kD bands were termed BDP1 and BDP2.
  • Example I shows that spectrin produces BDP's in the presence of Calpain I.
  • Example I also shows that staining after SDS-PAGE can be used to detect spectrin or BDP's in samples having the relatively high levels necessary for polypeptide bands to be visible to the naked eye upon exposure to stains such as Coomassie Blue.
  • the method of Example I is also well suited to relatively pure samples where the bands corresponding to the BDP's and intact spectrin can be easily identified. Samples having suitably high levels and purity of spectrin and/or BDP's are, for example, obtained from homogenized neural tissues after purification. See Davis and Bennett, supra.
  • a more sensitive method of detecting spectrin or BDP's, even in complex mixtures of polypeptides can advantageously be obtained by exposing the separated sample to antibodies reactive with spectrin or BDP's.
  • One assay which is suitable for this purpose has come to be known as a Western blot assay.
  • BDP's exhibit apparent stability towards further degradation, suggesting that antibodies directed against spectrin can recognize the BDP's in biological samples, such as tissues, fluids, etc. Both BDP's and intact spectrin can be recognized by antibodies directed against spectrin. Accordingly, anti-spectrin antibodies will detect both intact spectrin and BDP's when used in a Western blot assay.
  • the following example shows such a Western blot assay, using the gel obtained from Example I and anti-spectrin antibodies to detect the presence of both intact spectrin and BDP's.
  • Antibodies to brain spectrin were raised in rabbits using well known procedures (see, for example, Hurn, B.A.L. and Chantler, S.M., Meth. Enz. 10:104-135 (1988), which is incorporated herein by reference).
  • the anti-brain spectrin antibodies were purified from serum by brain spectrinsepharose affinity chromatography. Briefly, antibodies to brain spectrin were isolated from the serum by adsorption to brain spectrin coupled to ⁇ -amino hexanoic acid activated sepharose 4B (Sigma Chemical Co., St. Louis, Mo.). The specifically bound antibodies were then eluted in 0.2 M glycine, pH 2.8.
  • affinity purified antibodies were then equilibrated to pH 7.4 and frozen until use.
  • the antibodies were found to be reactive against BDP1 and BDP2 as well as to intact spectrin
  • the 150 kD bands which appeared upon exposure of spectrin to Calpain comprised polypeptides which were cross reactive with spectrin.
  • Purified brain spectrin was incubated as described in Example I. After SDS-PAGE, the proteins were electrophoretically transferred to a nitro-cellulose membrane using a Trans-Blotter (Bio-Rad, Richmond, Calif.) according to the manufacturer's recommendations for the transfer of high molecular weight proteins. The nitrocellulose sheets were incubated with anti-spectrin antibodies and the bound antibodies detected using an Immuno-Blot assay kit (also available from Bio-Rad) according to the manufacturer's directions.
  • Trans-Blotter Bio-Rad, Richmond, Calif.
  • anti-rabbit IgG Bio-Rad, Richmond, Calif. conjugated to alkaline phosphatase was used in a 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate system of detection according to the manufacturer's recommendations.
  • Affinity purified anti-spectrin antibody was diluted 1/750 (in a volume of 50 ml) and incubated overnight with the blot during the primary antibody step. The immunoreactivity of the bands is shown in FIG. 1B, lanes 1-3.
  • Lane 1 shows spectrin without exposure to Calpain
  • Lane 2 shows spectrin after treatment with 1 mM CaCl 2 and 1.8 ⁇ g/ml calpain I for 10 minutes
  • Lane 3 shows spectrin after treatment with 1 mM CaCl 2 and 3 ⁇ g/ml calpain I for 30 minutes. It can be seen that in the presence of Calpain, the degradation of spectrin produces BDP's, primarily BDP1 and BDP2.
  • a method for detecting cellular pathology comprising the steps of extracting a sample from a subject mammal and analyzing the sample for the presence of spectrin BDP's.
  • the analyzing step can involve an immunoassay using antibodies which recognize spectrin or stable breakdown products of spectrin.
  • the amount of BDP's is determined and this amount is compared to a basal level of BDP; an increase of BDP's being indicative of cellular death or degradation.
  • the basal level is the level of BDP's from healthy cells.
  • the basal level can be taken from a corresponding sample in a healthy subject mammal.
  • the basal level can be obtained from a sample from the same subject at a point in time prior to the insult.
  • a series of samples can be taken from a single subject over time and analyzed for the presence of BDP's, thereby advantageously providing an indication of the course of cellular death or degradation in the subject.
  • the level of BDP's is below the detection threshold of the assays described herein. Accordingly, the basal level against which the detected amount of BDP's are compared is often zero. Therefore, in many samples, the detection of any BDP's is indicative of cellular death or degradation.
  • the following example illustrates the establishment of a basal level for human CSF.
  • CSF samples are obtained from a healthy human subject. All CSF samples are concentrated by ultrafiltration. Two marker samples are also obtained to identify bands corresponding to intact spectrin and BDP's in completed gels.
  • the first marker sample is a sample of purified spectrin without exposure to Calpain as in Lane 1 of Example I to show the position of intact spectrin.
  • the other marker sample is of purified spectrin after exposure to Calpain as in Lane 3 of Example I to show the position of BDP's. Protein concentration of the samples and marker samples was determined by the method of Bradford, Anal Biochem. 72:248-254 (1976), the disclosure of which is hereby incorporated by reference.
  • the methods described herein can be used to measure BDP's in a variety of tissues and fluids because spectrin is found in a variety of tissues.
  • BDP's of spectrin have been observed in blood platelets (Fox, et al , Blood 69:537-545 (1987)) and intestinal brush border cells (Glenney, et al., PNAS 79:4002-4005 (1982)).
  • the following tissue taken from rats have been examined by the present inventors and others using the methods described herein and found to exhibit spectrin and BDP's: submandibular gland, brush border, testes, thymus, skeletal muscular, heart muscle, lung, liver, spleen, adrenal gland, kidney, brain.
  • humans, gerbils and mice have been determined by the present inventors and others to contain spectrin and BDP's, suggesting that spectrin and BDP's are common to all mammals.
  • Examples IV through VIII are provided, showing the detection of cellular death or degradation in the expected tissues.
  • Stereotaxically-placed unilateral electrolytic lesions of the entorhinal cortex were made in Sprague-Dawley rats. Animals were sacrificed after postoperative survival times of 0.2, 0.4, 1, 2, 4, 7, 14 and 27 days.
  • brains were rapidly dissected in ice-cold homogenization buffer consisting of 0.32 M sucrose, 10 mM Tris, 2 mM EDTA, 1 mM ethylene glycol bis ( ⁇ -amino-ethylester) N,N,N',N'-tetraacetic acid (EGTA), 100 ⁇ M leupeptin, 1 ⁇ g/ml N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), pH 7.4.
  • TPCK N-tosyl-L-phenylalanine chloromethyl ketone
  • the contralateral dentate gyrus tissue sample and the ipsilateral dentate gyrus tissue sample were each homogenized in 500 ⁇ l of dissection buffer. An aliquot of each was added to one-third volume of 3X SDS-PAGE sample buffer (consisting of 150 mM Tris-PO 4 , 6% SDS, 30% glycerol, 3.75 mM EDTA, 3% ⁇ -mercaptoethanol, pH 6.8) and placed in a 90° C. water bath for three minutes. The protein concentration of each homogenate was determined by the method of Bradford, supra. The concentration of proteins in each homogenate sample was then adjusted to 0.33 mg/ml with additional sample buffer.
  • 3X SDS-PAGE sample buffer consisting of 150 mM Tris-PO 4 , 6% SDS, 30% glycerol, 3.75 mM EDTA, 3% ⁇ -mercaptoethanol, pH 6.8
  • Antibodies were produced by the following method: For each rabbit, approximately 200 ⁇ g of purified brain spectrin was excised from SDS-polyacrylamide gels (after electrophoretic separation) and emulsified with Freund's complete adjuvant. Multiple subdermal injections were made and the procedure repeated again after two to four weeks, using Freund's incomplete adjuvant. After an additional two weeks, subcutaneous injections of an emulsion containing approximately 100 ⁇ g of spectrin were made. This procedure was repeated approximately one month later. Ten days following this series of injections, approximately 20 ml of blood was drawn from each rabbit and the serum collected after allowing the blood to clot overnight at 4° C.
  • Antibodies to brain spectrin were then affinity purified by adsorption to brain spectrin coupled to ⁇ -amino hexanoic acid activated sepharose 4B, as described in Example II.
  • the affinity purified antibodies were then equilibrated to pH 7.4 and frozen until use.
  • Example VI To determine the amount of spectrin immunoreactivity on the membrane of Example VI, the membrane was exposed to the antibody of Example VII as part of a Western Blot assay. Procedures for blocking, primary and secondary antibody incubations and color development were as described in Example II. Quantitation of the immunoreactive species was made using a soft laser scanning densitometer (Model #SLR504-XL, BioMed Instruments, Fullerton, Calif.). An integrator (Model 4270, Varian, Sunnyvale, Calif.) summed the amount of reaction product in each band and expressed them as a percentage of the total in that sample.
  • the anti-spectrin reactive species present in the contralateral (lane 1) and ipsilateral dentate gyrus (lane 2) two days after a unilateral entorhinal lesion are shown in FIG. 1A.
  • the homogenates of the ipsilateral dentate gyrus exhibited a marked increase in the amount of two peptides, termed BDP1 and BDP2, with apparent M r s of about 155,000 and 150,000 Daltons, respectively.
  • Example IV The procedures of Examples IV through VIII were repeated, allowing various lengths of time to elapse between the lesion and sacrifice of the animals of Example IV.
  • the time course of the changes in BDP1 and BDP2 in the dentate gyrus following a unilateral entorhinal cortex lesion is shown in FIG. 2.
  • BDP's are usually below the limit of detection in samples from unoperated animals.
  • a significant elevation of BDP's in the ipsilateral sample is evident as early as four hours post-lesion.
  • the increase is maximal two days after the lesion, where the BDP's represent 25% of the total immunoreactivity.
  • Examples IX through XI are provided in order to show that the methods utilized in Examples V through VIII have widespread utility in detecting cellular death or degradation. These examples show the detection of cellular death or degradation from a variety of causes and in a variety of cellular tissues through methods of the present invention. As such, these examples are intended to illustrate, not to limit the invention. While the procedures described herein, such as those of Examples V through VIII, are typical of those that might be used, other alternative procedures known to those skilled in the art can be alternatively employed.
  • Carotid arteries were clamped for 10 minutes to interrupt the principal blood flow to the cortex in each of two groups of eight Mongolian gerbils. Two control groups of gerbils were also analyzed. Samples of the CA1 hippocampal region and the cerebellum were taken at 4 hours after ischemia from one group of control gerbils and one group of test gerbils. Samples were also taken at 24 hours after ischemia from the second control and test groups of gerbils. The test gerbils showed elevated BDP's in the CA1 region compared to control animals, as shown in FIG. 5. The blood supply to the cerebellum was not interrupted and this structure showed no such increase. Analysis of BDP levels was as described in Example V-VIII.
  • the foregoing examples show that the methods of the present invention can advantageously be used to detect cellular death or degradation from a variety of causes in a variety of samples.
  • the present invention advantageously provides an additional method for the detection of cellular pathology without the necessity of separating the sample into BDP and intact spectrin.
  • This additional method is by means of an immunoassay to determine the presence of intact spectrin itself or spectrin immunoreactivity regardless of source. Therefore, in this embodiment of the invention, total spectrin immunoreactivity, including immunoreactivity to spectrin and to BDP's, can be measured.
  • an enzyme-linked immunosorbent assay (ELISA) for spectrin immunoreactivity in biological samples, such as tissue extracts, cerebrospinal fluid (CSF), or blood serum is provided.
  • a spectrin sample is immobilized to polystyrene microliter plates.
  • spectrin desorbs from conventional activated polystyrene plates after immobilization, resulting in an unexpected bell-shaped relationship between the amount of antibody bound and the amount of spectrin in the sample which is measured. While not wishing to be bound by any particular explanation for this unexpected result, it is believed that the desorbed spectrin forms polymers with still immobilized spectrin in the presence of accessory proteins present in the sample.
  • the spectrin polymers are believed to be more accessible to binding of anti-spectrin antibody.
  • spectrin in the sample is believed to bind to the plate through further polymerization of the spectrin.
  • polystyrene plates can be treated with glutaraldehyde prior to the immobilization of spectrin to the plates.
  • Glutaraldehyde forms covalent bonds to both the polystyrene of the plates and to the spectrin molecules.
  • buffers with conditions, including salinity and pH, unfavorable to the polymerization of spectrin has also been found to prevent the unexpected results.
  • High ion concentration has been found to inhibit formation of spectrin polymers, however, such concentrations also interfere with immunoreactivity. Addition of various other agents has also been found to prevent the unexpected results, including EGTA, sucrose and detergents.
  • buffers with a pH slightly greater than 7.0 in physiological saline with EGTA, sucrose and detergent is used.
  • Chaotropic salts, such as NaBr or KI, can also be used to inhibit formation of polymers.
  • a limiting amount of anti-spectrin antibody is added to each well along with the sample.
  • Spectrin in the sample competes for antibody with the spectrin immobilized to the plate.
  • the more spectrin in the sample the less antibody will bind to the spectrin immobilized to the plate.
  • the amount of antibody binding to the spectrin on the plate provides an indication of the amount of spectrin in the sample.
  • the amount of antibody can be detected by a colorimetric reaction as in a standard ELISA procedure, or can be detected in any known manner, such as through a radioimmune assay.
  • a spectrin preparation was prepared from rat brains by the method of Davis and Bennett (J. Biol. Chem. 258:7757-7766, 1983).
  • Antibodies to spectrin were prepared by subjecting the spectrin preparation to SDS-PAGE (see Seubert, et al., Synapse 1:20-34, 1987), excising the region of the gel containing the spectrin, homogenizing the gel and immunizing rabbits with the homogenized gel according to established procedures (see, e.g., Hurn and Chantler, Methods Enzymol. 70:104-135, 1980).
  • the lysine solution in each well was discarded and a sample of the unknown was placed in each well.
  • the absorbance at 405 nm of the wells containing unknown samples was also read and the concentration of spectrin determined by comparing the absorbance of the unknown wells to the standard curve.
  • the concentration of spectrin immunoreactivity correlates well with measurements of the same samples which are subjected to the Western Blot assay of Example VIII.
  • the following example demonstrates the correlation between the Western blot and the ELISA assay in neurodegenerating rats.
  • Rats were given intracerebral ventricle injections of 75 ng of kainate, a compound known to cause neurodegeneration within the hippocampus. A second set of rats were given equal volume injections of saline. The rats were allowed to recover for four days. The hippocampi were then removed and divided into four sections, rostral to caudal. Each section was analyzed using both the Western blot assay as in Example II, and using the ELISA assay of Example XII. Results are shown in FIG. 7.
  • FIG. 7 shows the left (rostral to caudal, 1-4) and right (5-8) hippocampi of a control animal and the left (9-12) and right (13-16) hippocampi from a kainate injected animal.
  • the top panel shows the Western Assay and the bottom panel shows the ELISA assay. It can be seen that the increase in the total amount of immunoreactivity measured by the ELISA correlates well with the increase in BDP's measured by the Western Blot assay.
  • One particular application of the ELISA of the present invention is the detection of spectrin or spectrin immunoreactivity in the cerebrospinal fluid (CSF) as an indication of neurodegenerative conditions, including subarachnoid hemorrhage, stroke, multiple infarction dementia, HIV-induced neuropathy and Alzheimer's Disease.
  • CSF cerebrospinal fluid
  • spectrin is present in isodermal cells lining the ventriculus and in particular in specific cells standing between the CSF and the blood, the number of such cells is very small compared to neural cells.
  • the death or degradation of glial cells, the support cells for neurons, would also be capable of contributing spectrin or BDP's to the CSF.
  • the death or degradation of the neural cells such cells support would follow shortly thereafter. Therefore, the vast majority of spectrin immunoreactivity found in the CSF would be indicative of breakdown of neural cells.
  • the cerebrospinal fluid of humans can be assayed either directly or after concentration using lyophilization or centrifugal ultrafiltration (using materials such as those sold under the trademarks "Centricon-10" or “Amicon”).
  • lyophilization or centrifugal ultrafiltration using materials such as those sold under the trademarks "Centricon-10" or “Amicon”.
  • the following example illustrates one typical method of the present invention for assaying human CSF for the presence of spectrin immunoreactivity.
  • Cerebrospinal fluid samples were obtained from 2 patients diagnosed as having unruptured aneurysms (group 1) and from 4 patients in which the aneurysm has burst producing subarachnoid hemorrhage (group 2). Two ml of each sample was lyophilized, resuspended in 100 ⁇ l water, and 10 ⁇ l of the resulting solution was analyzed for spectrin immunoreactivity using the ELISA assay of Example XII. Results are shown in FIG. 8. The CSF samples from the subarachnoid hemorrhage group all showed spectrin immunoreactivity while the unruptured aneurism group had no detectable spectrin immunoreactivity.
  • Example XIV the presence of detectable quantities of spectrin immunoreactivity in the CSF is indicative of cellular death or degeneration in neural tissue.
  • Total spectrin immunoreactivity was measured, using the ELISA method of Example XII, in CSF taken from a number of different patients suffering from a variety of conditions known to be associated with neurodegeneration. Results are shown in FIG. 9.
  • the first column of FIG. 9, labeled "ANEU" shows the results of two patients who had brain aneurisms detected and surgically corrected before the aneurisms burst, as in Group 1 of Example XIV. Thus, significant neurodegeneration would not be expected in these patients.
  • no spectrin immunoreactivity was found in CSF taken from these patients.
  • the data confirms that no spectrin immunoreactivity is detected in CSF in non-neurodegenerating mammals using the ELISA assay of the present invention.
  • SAH spectrin immunoreactivity measurements in CSF of 12 patients with subarachnoid hemorrhage who had had CSF drains installed.
  • the CSF from all 12 patients shows spectrin immunoreactivity, indicating that neurodegeneration has occurred.
  • AD Alzheimer's disease
  • the fourth column of FIG. 9 shows spectrin immunoreactivity in the CSF from one stroke victim. It can be seen that spectrin immunoreactivity is quite high in this patient, indicating significant neurodegeneration.
  • IVH The fifth column of FIG. 9, labeled "IVH” shows spectrin immunoreactivity from three premature infants suffering from intraventricular hemorrhage. Results show that two out of three of these patients show spectrin immunoreactivity in their CSF, indicating neurodegeneration.
  • the last column of FIG. 9 shows spectrin immunoreactivity in one victim of Pick's disease.
  • the results show high levels of spectrin immunoreactivity in this patient, indicative of the neurodegeneration which accompanies this disease
  • Pick's disease is clinically very difficult to distinguish from Alzheimer's disease.
  • Pick's can only be readily distinguished from Alzheimer's upon autopsy. It can be seen from the present data, that the Pick's sufferer had significantly higher levels of spectrin immunoreactivity than any of the Alzheimer's sufferers.
  • the present method will provide a diagnostic tool in distinguishing between these two diseases by the generally higher levels of spectrin immunoreactivity found in the CSF of Pick's patients.
  • the spectrin immunoreactivity detected in all of the foregoing examples is, of course, due to a large number of different antigenic epitopes. It is believed that after the proteolysis of spectrin to BDP's, additional or occult epitopes are exposed which are not present in intact spectrin. Thus, when performing the ELISA assay using polyclonal antibodies raised against BDP's, BDP's can give a stronger signal than the intact spectrin. In such assays, treating the spectrin in such a way to expose the occult epitopes, can also give a stronger signal than intact spectrin.
  • the anti-spectrin antibodies used in the Western Blot assays of examples II-XI were affinity purified using the affinity purification method described in Example II. This affinity purification step with intact spectrin, resulted in the purification of antibodies to epitopes of spectrin present and exposed in intact spectrin.
  • the raw serum contained at least two other classes of antibodies which react against spectrum.
  • One class of antibodies are to epitopes of spectrin not exposed in the intact tetrameres, but exposed in cleaved spectrin.
  • Another class of antibodies would be antibodies specific to spectrin-SDS complexes.
  • This class of antibodies is expected because the spectrin used to immunize the rabbits producing the antibodies in Example II was purified from SDS-PAGE, resulting in the formation of these SDS-spectrin complexes.
  • anti-spectrin antibodies raised against denatured rat spectrin react more efficiently with denatured spectrin than with intact spectrin, and that, therefore, occult epitopes exist and that antibodies against the occult epitopes can be used to distinguish native from denatured spectrin, the following example was performed.
  • the amount of spectrin immunoreactivity on the plate was determined by incubating plates with raw serum from immunized rabbits at 1:10,000 dilution in assay buffer (100 ⁇ l well) overnight at 4° C., rinsing the plates four times with wash buffer, and detecting bound antibody using the Vector ABC-AP kit as in Example XII. Results are shown in Table 1.
  • BDP BDP1 and BDP2 polypeptides visualized by Western blot and other spectrin fragments
  • altering the conditions of the solution to affect the solubility of the intact spectrin molecule By altering the pH, ionic strength, or other such factors, it is believed possible to solubilize the BDP's while precipitating the intact spectrin molecules.

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US07/558,700 US5118606A (en) 1988-09-02 1990-07-26 Methods for detecting cellular pathology by assaying spectrin and spectrin breakdown products
JP3512466A JP2824150B2 (ja) 1990-07-26 1991-07-11 スペクトリン又はその崩壊生成物の分析による細胞壊死検出
DE69113273T DE69113273T2 (de) 1990-07-26 1991-07-11 Zellnekrosnachweis durch prüfung von spektrin und seine abbauprodukte.
PCT/US1991/004910 WO1992001935A1 (en) 1990-07-26 1991-07-11 Cell necrosis detection through assays for spectrin and breakdown products thereof
AT91913322T ATE128233T1 (de) 1990-07-26 1991-07-11 Zellnekrosnachweis durch prüfung von spektrin und seine abbauprodukte.
ES91913322T ES2077863T3 (es) 1990-07-26 1991-07-11 Deteccion de necrosis celular por medio de analisis de deteccion de la espectrina y de los productos de descomposicion de esta ultima.
DK91913322.3T DK0540591T3 (da) 1990-07-26 1991-07-11 Cellenekrosepåvisning ved prøvning for spektrin og dets nedbrydningsprodukter
EP91913322A EP0540591B1 (de) 1990-07-26 1991-07-11 Zellnekrosnachweis durch prüfung von spektrin und seine abbauprodukte
CA002088050A CA2088050A1 (en) 1990-07-26 1991-07-11 Cell necrosis detection through assays for spectrum and breakdown products thereof
GR950402366T GR3017459T3 (en) 1990-07-26 1995-09-21 Cell necrosis detection through assays for spectrin and breakdown products thereof.
JP10130877A JPH1130615A (ja) 1990-07-26 1998-05-13 スペクトリン又はその崩壊生成物の分析による細胞壊死検出

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JP2824150B2 (ja) 1998-11-11
DK0540591T3 (da) 1996-02-05
JPH1130615A (ja) 1999-02-02
ES2077863T3 (es) 1995-12-01
JPH05508919A (ja) 1993-12-09
EP0540591B1 (de) 1995-09-20
EP0540591A1 (de) 1993-05-12
ATE128233T1 (de) 1995-10-15
CA2088050A1 (en) 1992-01-27
WO1992001935A1 (en) 1992-02-06
DE69113273D1 (de) 1995-10-26
DE69113273T2 (de) 1996-05-15
GR3017459T3 (en) 1995-12-31

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